1. Field of the Invention
The present invention relates to the development of monomers of nucleic acid analogs and their oligomers. More specifically, the present invention relates to novel nucleic acid analog monomers and oligomers which use a piperazine ring in place of the ribose or deoxyribose sugar and to methods of synthesizing such monomers and oligomers.
2. Description of Related Art
Nucleotides and nucleosides are important molecules that participate in many vital cellular functions. Nucleotides serve as cellular signaling molecules, carriers of chemical energy, and building blocks of nucleic acids. See Alberts, et al., Molecular Biology of the Cell, 3d ed., (1994). Nucleotides also combine with other compounds to form enzymes.
Structurally, nucleotides are composed of a purine or pyrimidine base attached to a ribose or deoxyribose sugar with, in some cases, one or more phosphate groups joined to the sugar by ester linkages. Nucleotides lacking the phosphate groups are called nucleosides. Energy is stored in nucleotides as bond energy by enzymes that attach the phosphate groups to them in a reaction called phosphorylation. These enzymes are commonly known as “kinases,” an abbreviation of the term “phosphokinase.”
Nucleotides may be found alone as monomers, but are also often found as oligonucleotides-polymeric sequences of nucleotides joined together. Oligonucleotides have been shown to be useful in many applications, including as PCR primers, as probes in DNA cloning, in mutagenesis techniques, and in gene expression. In order to extend the functions of these useful polymers, efforts have been made to construct oligonucleotides using artificial nucleotides or molecules analogous to nucleotides. Such oligonucleotides are often referred to as oligonucleotide analogs.
Oligonucleotide analogs can be configured to bind DNA or RNA in a sequence specific manner. Agrawal, Antisense Therapeutics, (1996); de Mesmaeker et al., Acc. Chem. Res., 28:366 (1995); and Nielsen Adv. DNA Sequence Specific Agents, 3, 267-278, (1998). Such oligonucleotides are referred to as “antisense” when they are complementary to a sequence of mRNA. Thus when added to a mixture containing the mRNA, they bind to the mRNA, deactivating it. In contrast, they are referred to as “antigene” when they are complementary to the sequence of a gene, and are thus capable of binding to the gene. Such binding generally prevents further transcription of the gene.
Because oligonucleotides and their analogs have such useful functions, they have become the focus of research seeking novel approaches for modulating genetic diseases using drugs. Rhodes and James, AIDS 5, 145, (1991); Li et al., J. Virol. 76, 6882, (1993); Liesziewicz et al., Proc. Natl. Acad. Sci. U.S.A., 90, 3860, (1993). They have also garnered attention as research tools for use in probing gene function, genomic structure, and RNA folding. Using oligonucleotide analogs as a targetable knock-out tool holds promise for extracting important information from available genetic information. Mologni et al., Nucleic Acids Res., 26(8), 1934-1938, (1998); Good and Nielsen Nat. Biotechnol., 16(4), 355-358, (1998); Good et al., Nielsen Proc. Natl. Acad. Sci. U.S.A., 95(5), 2073-2076, (1998).
Hexose nucleotides and peptide-linked oligonucleotides have become a prime focus of research as possible precursors of RNA/DNA since to date no prebiotic ribose or pentose nucleotide phosphate syntheses have been demonstrated. Pitsch et al., Helv. Chim. Acta., 76(6):2161-2183 (1993); Orgel, NATO ASI Ser., Ser. A, 169:215-224 (1989); Bohler, et al., Nature, 376(6541):578-581; and Pitsch et al., Origins Live Evol. Biosphere, 25(4):297-334 (1995). Aside from this, the instability of DNA/RNA renders it unfit for many medical and research applications that require an information encoding system with greater shelf-stability.
The most commonly used nucleotide analogs include phosphothioates, methylphosphonates, and peptide nucleic acids. Miller et al., Biochemistry, 20:1878 (1981); Heinemann et al., Nucleic Acids Res., 19:42 (1991); Jayaraman et al., Proc. Natl. Acad. Sci. U.S.A., 78:1537 (1981); Wittung et al., Nature, 368:561 (1994); Nielsen et al., Science, 254:1497 (1991); and Egholm et al., Nature, 365:566 (1993). Phosphothioates and methylphosphonates are successfully used in part because they are not subject to the rapid nuclease-mediated biodegradation that renders DNA and RNA unstable. These analogs are generally found as heterochiral mixtures, and are difficult to obtain in an enantiomerically pure form. Stec & Wilk, Angew. Chem., 106:747 (1994). Disadvantages of these analogs include the fact that they are unspecific in their binding to proteins, and that they are often cytotoxic. Sarmiento et al., Antisense Res. and Develop., 2:99 (1994).
Peptide nucleic acids (“PNAs”) are another commonly used form of nucleic acid analogs. In structure, PNAs are amide-linked and non-chiral. They have a strong binding affinity to natural oligonucleotides. Nielsen, Biophys. Chem., 68(1-3):103-108 (1997). Another reason for their current widespread use is their ability to be synthesized through peptide solid phase synthesis. Christensen, et al., J. Pept. Sci., 1(3):185-183 (1995).
Newer PNAs have been modified to be chiral, use side chains, improve solubility, induce predictable binding preference for antiparallel double strand orientation, and to influence helical orientation on PNA binding. Still other new conformationally restricted piperidinone PNA adenine monomers have been developed and used in duplex formation. Nielsen et al., J. Chem. Soc., Perkin Trans., 1:2757-2763 (2001).
These currently used nucleotide analogs are limited in use, however, due to several of their inherent properties. First, uncharged linear oligonucleotides decrease in solubility in purine-rich sequences and as they increase in length. Second, their physicochemical properties are often limited by the base composition of the sequence. Third, they exhibit strong tendencies to self-aggregate, and are often characterized by low intracellular bioavailability. Fourth, all commonly-known uncharged nucleotide analogs require delivery through cell and nuclear membranes for antigene use, and require high intracellular concentrations for antisense use. Fifth, most of the known charged nucleotide analogs are incapable of inducing digestion of mRNA by RNAse H, a drug target that is promising in its potential use to regulate mRNA translation by blocking an undesired sequence's expression.
From the foregoing, it will be appreciated that it would be a significant advancement in the art to provide novel nucleic acid analog monomers, their oligomers, and methods for synthesizing them. It would be a further advancement in the art to provide novel nucleic acid analogs that exhibit improved solubility properties. It would be yet a further improvement in the art to provide novel nucleic acid analogs which exhibited less tendency to self-aggregate than those known in the art. Additionally, it would be an improvement in the art to provide nucleic acid analogs with improved in vitro bioavailability and which is usable with simple drug delivery methods. The nucleic acid analogs of the invention present additional functionality beyond complementary base recognition and lead to novel biochemical and biological function. Finally, it would be an improvement in the art to provide nucleic acid analogs with the ability to induce RNAse digestion of mRNA by inducing the action of RNAse H. Such compounds and methods are disclosed herein.